The broad goal of this application is to define the physiological relevance and the structural basis of the regulation of CFTR Cl- channels by syntaxin 1A. Syntaxin 1A is a membrane protein that is expressed in colonic epithelial cells where it localizes at or near the apical cell surface. Syntaxin 1A physically interacts with CFTR and negatively modulates CFTR C1- currents when these molecules are co- expressed in Xenopus oocytes. Our unpublished results indicate that the physical interaction between syntaxin 1A and CFTR is inhibited by PKA phosphorylation and by n-Sec1, a syntaxin-binding protein that reverses the negative modulation of CFTR by syntaxin 1A. We hypothesize that syntaxin 1A fine tunes CFTR C1- current activity in response to physiological clues (e.g., PKA activation). The specific aims of this proposal are as follows. First, we will test the hypothesis that the CFTR-syntaxin 1A interaction is regulated by physiologically relevant factors; namely PKA, PKC and n-Sec1. PKC is of interest because it phosphorylates n-Sec1 and inhibits its ability to bind syntaxin 1A; thus, PKC activation may release syntaxin 1A from n-Sec1 and thereby control the availability of syntaxin 1A for CFTR. Second, we will define the minimal domains of CFTR and syntxin 1A that are required for binding and test the functional activities of mutants that are defective at binding. One of our long-term goals is to generate functionally active CFTR mutants that cannot be negatively modulated by syntaxin 1A in vivo. Third, we will test the hypothesis that CFTR and syntaxin 1A co- reside in a molecular complex at the apical surfaces of colonic epithelial cells. We will determine if syntaxin 1A co-immunoprecipitates with surface-labeled CFTR. We will also determine if CFTR resides with syntaxin 1A in a multimeric complex that includes additional candidate regulators of C1- current activity. The results of our studies should provide new insights into the regulation of CFTR function in epithelial tissues.